Microbial proteases are among the most important hydrolytic enzymes and find applications in various industrial sectors, such as detergents, food, leather, pharmaceuticals, diagnostics, waste management and silver recovery. Microbial extracellular proteases account for a major part, more than one third, of the total worldwide industrial enzyme sales (Chemy and Fidantsef, 2003). Approximately 90% of the commercial proteases are detergent enzymes (Gupta et al., 2002). The commercial detergent preparations currently in use comprise the naturally occurring alkaline serine proteases (EC 3.4.21) of the subtilisin family or subtilisins, originating from Bacillus species, or are recombinant protease preparations thereof (Maurer, 2004).
Examples of commercial proteases are such as subtilisin Carlsberg (Alcalase®), subtilisin 309 (Savinase®), Subtilisin 147 (Esperase®), Kannase®, EverlaseD, Ovozyme®, and the cold-wash protease Polarzyme® (Novozymes A/S, DK); Purafect®, Purafect® Ox, Purafect® Prime and Properase® (Genencor Int., Inc., USA); and the BLAP S and X series (Henkel, Del.).
Several alkaline serine proteases and genes encoding these enzymes have also been isolated from eukaryotic organisms, including yeast and filamentous fungi. U.S. Pat. No. 3,652,399 and EP 519229 (Takeda Chemical Industries, Ltd., JP) disclose an alkaline protease from the genus Fusarium (asexual state, teleomorph) or Gibberella, (sexual state, anamorph) particularly from Fusarium sp. S-19-5 (ATCC 20192, IFO 8884), F. oxysporum f. sp. lini (IFO 5880) or G. saubinetti (ATCC 20193, IFO6608), useful in the formulation of detergent and other cleanser compositions. WO 88/03946 and WO 89/04361 (Novo Industri A/S, DK) disclose an enzymatic detergent additive and a detergent composition comprising a protease and a lipase, wherein the fungal protease is derived from Fusarium, particularly F. oxysporum or F. solani. WO1994025583 (NovoNordisk A/S, DK) discloses an active trypsin-like protease enzyme derivable from a Fusarium species, in particular a strain of F. oxysporum (DSM 2672), and the DNA sequence encoding the same. The amino acid sequence of a novel protease deriving from Fusarium sp. BLB (FERM BP-10493) is disclosed in WO 2006101140 (SODX Co. Ltd, Nakamura). Use of F. equiseti and other fungi in reducing organic matter in waste waters is disclosed in the EP 1464626 patent application (Biovitis S.A., FR). The amino acid and nucleotide sequences of the serine proteases from F. equiseti and F. acuminatum have been disclosed in FI20095497 and FI20095499, respectively (AB Enzymes Oy, FI). The amino acid and nucleotide sequences of the serine protease derived from several Trichoderma species have been disclosed e.g. in WO2008045148A (Catalyst Biosciences, Inc., U.S.A.), WO96/018722A (Centro de Investigacion y de Estudios Avanzados del I.P.N., MX) and WO98/020116A (Novo Nordisk A/S, DK). Also, alkaline proteases from fungal species such as Tritirachium and Conidiobolus have been reported (reviewed in Anwar and Saleemuddin, 1998).
Different methods have been used for improving the stability of the industrial serine proteases. WO 92/03529 (NovoNordisk A/S, DK) discloses detergent compositions comprising a reversible protease inhibitor of the peptide or protein type. In one preferred embodiment the protease is a subtilisin, preferably derived from Bacillus and the inhibitor is a subtilisin inhibitor of family III, VI or VII. The liquid detergent compositions comprising proteases often include protease inhibitors such as boric acid with or without polyols to inhibit the autocatalytic activity of proteases. One of such inhibitors is 4-formyl phenyl boronic acid (4-FPBA) disclosed in US0120649 (Novozymes A/S, DK). EP0352244A2 (NovoNordisk A/S, DK) discloses improvement of stability of Bacillus derived enzymes using amphoteric compounds, such as surfactants.
Variants of the natural serine proteases with improved catalytic efficiency and/or better stability towards temperature, oxidizing agents and different washing conditions have been developed through site-directed and/or random mutagenesis. Most of the work has been carried out with Bacillus derived subtilisins. Replacement of one or more amino acid residues of B. amyloliquefaciens subtilisin is disclosed in WO98/55634 and WO99/20727 (Procter & Gamble Co., Genencor Int., Inc., U.S.A.) and in WO99/20770, US20090011489A and EP1025241B2 (Genencor Int., Inc., U.S.A.). Protease variants of B. lentus subtilisin with improved wash performance are disclosed in WO2003/062381 and EP1523553B1 (Genencor Int., Inc., U.S.A.). The substitutions of R170S-A1R, R170S-G61R, R170S-N216R or R170S-0100R do not change the net electrostatic charge of the variant when compared to the precurcor protease. Multiply-substituted variants of B. lentus protease with altered net charge, resulting in improved efficacy at low, medium or high detergent concentrations is disclosed in EP 1612271 A2 (Genencor Int., Inc. U.S.A.).
EP2138574A2 (Novozymes A/S, DK) discloses subtilase variants including insertion, substitution or deletion of an amino acid in one or more positions of the amino acid sequence of subtilisin BPN′. US2009/0203111A1 (Novozymes A/S, DK) discloses JP170 and BPN′ variants having altered properties and methods for their production. Bacillus sp. subtilisin variant useful in dishwashing detergent is disclosed in US20100152088A (Danisco US, Inc., U.S.A.).
Variants of fungal serine proteases have been prepared for trypsin-like proteases. WO95/030743A1 (NovoNordisk A/S, DK) discloses variants of a trypsin-like Fusarium protease, in which a naturally occurring amino acid residue (other than proline) is substituted with a proline residue at one or more positions which positions are not located in regions in which the protease is characterized by possessing alpha-helical or beta-sheet structure. The variants have improved proteolytic stability and are less susceptible to oxidation as compared to the parent protease. EP1546318B (Novozymes Inc., U.S.A.) discloses trypsin variants of Fusarium oxysporum comprising substitutions, insertions or deletions of one or more amino acids of the precursor protease.
Despite the fact that numerous patent publications, reviews and articles have been published, in which serine proteases from various microorganisms, for example, the low temperature alkaline proteases from actinomycete (Nocardiopsis dassonvillei) and fungal (Paecilomyces marquandii) microorganisms are disclosed, e.g. in EP 0290567 and EP 0290569 (Novo Nordisk A/S, DK), there is still a great need for alternative serine proteases, which are suitable for and effective in modifying, degrading and removing proteinaceous materials of different stains, particularly in low or moderate temperature ranges and which are stable in the presence of detergents with highly varying properties. Due to autocatalytic property of serine proteases, the stability during storage is also very important.
It is also desirable that the serine protease can be produced in high amounts, and can be cost-effectively down-stream processed, by easy separation from fermentation broth and mycelia.